Black rice diet alleviates colorectal cancer development through modulating tryptophan metabolism and activating AHR pathway

Abstract

Consumption of dietary fiber and anthocyanin has been linked to a lower incidence of colorectal cancer (CRC). This study scrutinizes the potential antitumorigenic attributes of a black rice diet (BRD), abundantly rich in dietary fiber and anthocyanin. Our results demonstrate notable antitumorigenic effects in mice on BRD, indicated by a reduction in both the size and number of intestinal tumors and a consequent extension in life span, compared to control diet-fed counterparts. Furthermore, fecal transplants from BRD-fed mice to germ-free mice led to a decrease in colonic cell proliferation, coupled with maintained integrity of the intestinal barrier. The BRD was associated with significant shifts in gut microbiota composition, specifically an augmentation in probiotic strains Bacteroides uniformis and Lactobacillus. Noteworthy changes in gut metabolites were also documented, including the upregulation of indole-3-lactic acid and indole. These metabolites have been identified to stimulate the intestinal aryl hydrocarbon receptor pathway, inhibiting CRC cell proliferation and colorectal tumorigenesis. In summary, these findings propose that a BRD may modulate the progression of intestinal tumors by fostering protective gut microbiota and metabolite profiles. The study accentuates the potential health advantages of whole-grain foods, emphasizing the potential utility of black rice in promoting health.

Keywords: black rice diet; colorectal cancer; gut metabolites; gut microbiome.

Figure 1

BRD against intestinal tumorigenesis in Apc ^Min/+ mouse model. (A) Experimental design for Apc ^Min/+ CRC mouse model and WT mice subjected to either a black rice‐fed diet or a control‐fed diet. (B) Enhanced survival observed in BRD‐fed mice (n = 28 per group) compared with CD‐fed mice (n = 35 per group). (C) Body weight of BRD‐fed and CD‐fed mice before killing (n = 9 per group). (D) Representative colon image at the time of killing. Tumor number and tumor volume in BRD‐fed and CD‐fed mice. The structures indicated by the red arrows are tumors. (E) H&E staining for pathological diagnosis of mice colons. Quantitative analysis of the pathological score employed the following criteria: 0, normal; 1, low‐grade dysplasia; 2, high‐grade dysplasia; and 3, carcinoma. (F) IHC staining for Ki‐67 in mice colons, accompanied by a quantitative analysis of the Ki‐67 index. (G) Expression levels of proliferating cell nuclear antigen (PCNA) protein in colon tissues of BRD‐fed and CD‐fed mice using western blot analysis with quantitative analysis. (H) Lipopolysaccharide (LPS) concentration in serum of BRD‐fed and CD‐fed mice in an Apc ^Min/+ model. The relative protein levels are normalized to those of the control β‐actin. (I) Representative images of intercellular junctions captured by transmission electron microscopy. The structures highlighted by the red arrows are the focal points. (J) The number of colon goblet cells assessed by PAS staining. (K–N) IHC for the distribution of adhesion molecules ZO‐1, Claudin‐3, and occludin with quantitative analysis in colon tissues of BRD‐fed and CD‐fed mice. (O, P) Anti‐inflammatory interleukin (IL)‐4 and IL‐10 concentrations and (Q, R) pro‐inflammatory TNF‐α and IL‐6 concentrations in serum of BRD‐fed and CD‐fed mice in an Apc ^Min/+ model. BRD, black rice diet; CD, control diet; CRC, colorectal cancer; H&E, hematoxylin and eosin; HGD, high‐grade dysplasia; IHC, immunochemistry; IL‐6, interleukin‐6; LGD, low‐grade dysplasia; PAS, periodic acid–Schiff; TNF‐α, tumor necrosis factor‐α; WT, wild type. *p < 0.05, **p < 0.01, N.S., no significant. Dot plots reflect data points from independent experiments.

Figure 2

Black rice diet (BRD)‐modulated gut microbiota inhibits gut barrier dysfunction in germ‐free mice. (A) Experimental design for stools were transplanted from BRD‐fed mice and control diet‐fed mice to germ‐free mice (n = 7 per group) under CD. (B) H&E staining for pathological diagnosis of mice colons. Quantitative analysis of the pathological score employed the following criteria: 0, normal; 1, low‐grade dysplasia; 2, high‐grade dysplasia; and 3, carcinoma. (C) IHC staining for Ki‐67 and quantitative analysis of Ki‐67 index of GF‐CD and GF‐RBD mice colons. (D) Expression levels of cell proliferating indicating protein proliferating cell nuclear antigen (PCNA) in colon tissues of GF‐CD and GF‐BRD mice. The relative protein levels are normalized to those of the control β‐actin. (E) The number of colon goblet cells assessed by periodic acid–Schiff (PAS) staining. (F) Lipopolysaccharide (LPS) concentration in serum of GF‐CD and GF‐BRD mice. (G) Representative images of intercellular junctions captured by transmission electron microscopy. The structures highlighted by the red arrows are the focal points. (H–K) IHC for the distribution of adhesion molecules ZO‐1, claudin‐3, and occludin with quantitative analysis in colon tissues of GF‐CD and GF‐BRD mice. (L, M) Anti‐inflammatory IL‐4 and IL‐10 concentrations and (N, O) pro‐inflammatory tumor necrosis factor‐α (TNF‐α) and IL‐6 concentrations in serum of GF‐CD and GF‐BRD mice. GF‐BRD, germ‐free mice gavaged with fecal samples of BRD‐fed mice; GF‐CD, germ‐free mice gavaged with fecal samples of CD‐fed mice; H&E, hematoxylin and eosin; HGD, high‐grade dysplasia; IHC, immunochemistry; LGD, low‐grade dysplasia. *p < 0.05, **p < 0.01, N.S., no significant. Dot plots reflect data points from independent experiments.

Figure 3

The black rice diet (BRD) altered gut microbial composition and increased the abundance of beneficial bacteria in the Apc ^Min/+ model. (A) Alpha‐diversity analysis using the Simpson index in wild‐type mice, control diet (CD)‐fed, or BRD‐fed mice at 14 weeks (WT_14, n = 38 per group, CD_14, n = 15 per group, and BRD_14, n = 14 per group) and at 22 weeks (WT_22, n = 14 per group, CD_22, n = 22 per group, and BRD_22, n = 15 per group). (B) β‐diversity analysis using the Bray–Curtis distance. (C) Identification of marker microbes differentiating groups between black rice and CDs or between the wild type (WT) and control (p < 0.05, LDA > 2). (D) Relative abundance of Lactobacillus johnsonii and Bacteroides uniformis. (E) Co‐occurrence analysis: Spearman correlation coefficients between microbes. Different colors represent different bacteria. Red lines indicate positive correlations, blue lines indicate negative correlations, and gray lines indicate no correlation. (F) Relative abundance of Bacteroides uniformis in six cohort data sets via meta‐analysis. (G) Cell growth curves of CRC cell lines HCT116 and SW620 treated with B. uniformis and E. coli (as a negative control). BRD, black rice diet; CD, control diet; CRC, colorectal cancer; LDA, linear discriminant analysis. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, N.S., no significant. Dot plots reflect data points from independent experiments.

Figure 4

BRD altered intestinal feces metabolite composition and enhanced tryptophan metabolism pathway in the Apc ^Min/+ model. (A) principal component analysis (PCA) plot for gut metabolomics analysis in wild‐type (WT), control diet (CD)‐fed, or BRD‐fed mice at 14 weeks (WT_14, n = 14 per group, CD_14, n = 16 per group, and BRD_14, n = 15 per group) and at 22 weeks (WT_22, n = 14 per group, CD_22, n = 16 per group, and BRD_22, n = 15 per group). (B) Identification of marker metabolites differentiating black rice and CDs or between WT and control. (C) Analysis of differential metabolite traceability: percentage of host (4.08%), microbial (7.14%), shared (4.08%), food‐related (14.29%), and other (70.41%) sources. (D) Enrichment analysis of the co‐upregulated metabolites from BRD‐fed Apc ^Min/+ and CD‐fed WT mice. Tryptophan metabolic pathways are highlighted in red. (E) Three metabolites involved in the tryptophan metabolic pathway: tryptophan, indole, and indole‐3‐lactic acid. (F) Correlation analysis of Lactobacillus johnsonii and Bacteroides uniformis with indole and indole‐3‐lactic acid. Spearman correlation coefficient R and p values were marked. BRD, black rice diet; CD, control diet. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, N.S., no significant. Dot plots reflect data points from independent experiments.

Figure 5

Indole‐3‐lactic acid activates host AHR to inhibit cell proliferation and cell junction impairment. (A) Cell growth curves of colorectal cancer (CRC) cell line HCT116 cells treated with indole‐3‐lactic acid and a vehicle (as negative control). (B) HCT116 cells treated with or without indole‐3‐lactic acid were stained with propidium iodide (PI) and analyzed using flow cytometry. (C) Expression levels of cell proliferation and cell cycle–associated proteins proliferating cell nuclear antigen (PCNA) and cyclin D1, in HCT116 cells treated with indole‐3‐lactic acid and a vehicle. (D) Expression levels of gut barrier function‐associated proteins occludin and claudin‐3 in HCT116 cells treated with or without indole‐3‐lactic acid. (E) The differentially expressed patterns of AHR core targets in colon tissues of wild‐type (WT), black rice diet (BRD)‐fed, and CD‐fed mice and KEGG pathways from core target enrichment in Cluster2 and Cluster3. (F) qRT‐PCR results showed that BRD promoted AHR downstream target gene expression. The relative RNA levels are normalized to those of the control β‐actin. (G) Cell growth curves of CRC cell line HCT116 colon cell lines treated with DMSO, indole‐3‐lactic acid, DMSO + CH‐223191 (a potent and specific antagonist of AHR), and indole‐3‐lactic acid + CH‐223191. (H) HCT116 cells treated with DMSO, indole‐3‐lactic acid, DMSO + CH‐223191, and indole‐3‐lactic acid + CH‐223191 were stained with PI and analyzed using flow cytometry. (I) Expression levels of gut barrier function‐associated proteins occludin and claudin‐3 in HCT116 cell lines treated with DMSO, indole‐3‐lactic acid, DMSO + CH‐223191, and indole‐3‐lactic acid + CH‐223191. The relative protein levels are normalized to those of the control β‐actin. AHR, aryl hydrocarbon receptor; DMSO, dimethyl sulfoxide; KEGG, kyoto encyclopedia of genes and genomes; qRT‐PCR, quantitative real‐time polymerase chain reaction. Data are expressed as mean ± SD. *p  < 0.05, **p  < 0.01, N.S., no significant.

Figure 6

Black rice diet (BRD) alleviates colorectal cancer (CRC) development through modulating tryptophan metabolism and upregulating aryl hydrocarbon receptor (AHR) pathway. The BRD may attenuate CRC tumor development in CRC mouse models by promoting the abundance of protective gut microbiota and metabolites, as well as activating the host intestinal AHR pathway.